a-Synuclein is a small protein enriched in presynaptic nerve terminals throughout the brain. Though predominantly cytosolic, the protein also has a high affinity for phospholipid membranes. This membrane- binding ability is most likely essential for the protein's normal function, which consists of modulating the release of synaptic vesicles. Neuropathological and genetic data suggest that aggregated (oligomeric) species of a-synuclein are associatedwith neurodegeneration in Parkinson's disease (PD). The long-term objectives of the proposed research are to identify aggregated forms of a-synuclein that are valid drug targets in PD and to characterize the molecular interactions that lead to the formation of these aggregates in diseased neurons. The work described in this application is focused on the problem of whether phospholipid membranes play a role in the formation of neurotoxic a-synuclein aggregates. It is hypothesized that membranes act as a 'platform'to trigger the formation of harmful a-synuclein oligomers. The project will address this hypothesis with the following specific aims: (1) to determine whether membrane binding promotes the formation of (3-sheet-rich a-synuclein aggregates in test-tube models;(2) to determine whether a-synuclein forms membrane-bound, potentially toxic aggregates in eukaryotic cells;(3) to examine the effects of a-synuclein oxidation on the formation of membrane-bound aggregates. The aggregation of the protein on supported lipid bilayers will be monitored by total internal reflection fluorescence microscopy and attenuated total reflection Fourier transform infrared spectroscopy. The formation of a-synuclein oligomers on membranes will also be monitored in test-tube models, yeast, or doparnine neurons via (i) differential centrifugation combined with Western blot analysis;(ii) fluorescence measurements, using an environment- sensitive fluorophore;and (iii) fluorescence lifetime imaging microscopy. Cell viability studies will be conducted to determine whether the formation of membrane-bound aggregates correlates with the induction of toxicity in doparnine neurons. These methods will also be used to determine whether the oxidation of a- synuclein affects the formation of membrane-bound a-synuclein oligomers in test-tube models or eukaryotic cells. The results of these studies will provide clues as to whether the aggregation of a-synuclein on membrane surfaces is linked to neurotoxicity in PD. Evidence that membrane-bound a-synuclein oligomers are valid drug targets would facilitate the development of screening assays to identify novel therapeutics.